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In vitro perforation of human epithelial carcinoma cell with antibody-conjugated biodegradable microspheres illuminated by a single 80 femtosecond near-infrared laser pulse.

Terakawa M, Tsunoi Y, Mitsuhashi T - Int J Nanomedicine (2012)

Bottom Line: A polylactic acid (PLA) sphere, a biodegradable polymer, was used.Fluorescein isothiocyanate (FITC)-dextran and short interfering RNA were delivered into many human epithelial carcinoma cells (A431 cells) by applying a single 80 fs laser pulse in the presence of antibody-conjugated PLA microspheres.Perforation by biodegradable spheres compared with other particles has the potential to be a much safer phototherapy and drug delivery method for patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Electronics and Electrical Engineering, Keio University, Yokohama, Kanagawa, Japan. terakawa@elec.keio.ac.jp

ABSTRACT
Pulsed laser interaction with small metallic and dielectric particles has been receiving attention as a method of drug delivery to many cells. However, most of the particles are attended by many risks, which are mainly dependent upon particle size. Unlike other widely used particles, biodegradable particles have advantages of being broken down and eliminated by innate metabolic processes. In this paper, the perforation of cell membrane by a focused spot with transparent biodegradable microspheres excited by a single 800 nm, 80 fs laser pulse is demonstrated. A polylactic acid (PLA) sphere, a biodegradable polymer, was used. Fluorescein isothiocyanate (FITC)-dextran and short interfering RNA were delivered into many human epithelial carcinoma cells (A431 cells) by applying a single 80 fs laser pulse in the presence of antibody-conjugated PLA microspheres. The focused intensity was also simulated by the three-dimensional finite-difference time-domain method. Perforation by biodegradable spheres compared with other particles has the potential to be a much safer phototherapy and drug delivery method for patients. The present method can open a new avenue, which is considered an efficient adherent for the selective perforation of cells which express the specific antigen on the cell membrane.

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Related in: MedlinePlus

Fluorescence (A and C) and phase contrast (B and D) images of A431 cells perforated by using antibody-conjugated polylactic acid spheres irradiated by a single fs laser pulse at 1.06 J/cm2 in the presence of fluorescein isothiocyanate-dextran (A and B) and Alexa Fluor-labeled small interfering RNA (C and D).Note: Dashed circles (300 μm diameter) indicate the laser irradiated area. Abbreviation: fs, femtosecond.
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f4-ijn-7-2653: Fluorescence (A and C) and phase contrast (B and D) images of A431 cells perforated by using antibody-conjugated polylactic acid spheres irradiated by a single fs laser pulse at 1.06 J/cm2 in the presence of fluorescein isothiocyanate-dextran (A and B) and Alexa Fluor-labeled small interfering RNA (C and D).Note: Dashed circles (300 μm diameter) indicate the laser irradiated area. Abbreviation: fs, femtosecond.

Mentions: Figure 4 shows fluorescence images (A and C) and phase contrast images (B and D) of the cells after the 80 fs laser illumination at the laser fluence of 1.06 J/cm2, corresponding to the peak intensity of 1.29 × 1014 W/cm2 under the sphere. The corresponding incident energy to the fs laser illuminated area (300 μm in diameter) is 0.75 mJ. Figure 4A and B show experimental results obtained with the FITC-dextran, while Figure 4C and D show those obtained with the siRNA. The average number of cells in the fs laser illuminated area was 221. Many cells in the illuminated area showed fluorescence, demonstrating the increase in the cell membrane permeability. The average perforation efficiency, which was defined as the fraction of cells in the irradiated area that took up exogenous molecules, evaluated by using FITC-dextran at this laser fluence, was 26.4% ± 7.5% (N = 5), while that evaluated by using siRNA was 34.9% ± 6.5% (N = 7). No statistically significant difference was observed in the perforation efficiencies with FITC-dextran and siRNA (P > 0.05 in nonparametric Mann-Whitney test). The perforation efficiency obtained by using PLA spheres is comparable to that shown in a previous study using PS sphere,23 demonstrating the effectiveness and applicability of biodegradable polymer for the perforation. As shown in Figure 2H, the PLA sphere of 2000 nm diameter works as a microlens and the focused intensity (far field) is kept high for distances longer than a few micrometers under the sphere, reaching a peak value at 870 nm under the sphere. The distance between the antibody-conjugated sphere and the cell membrane is several tens of nanometers. The long-focused zone of the sphere conjugated to the top surface of cell membrane is considered to be in the cytoplasm. Therefore, it is highly probable that the mechanism for perforation is not only due to the ablation of cell membrane.


In vitro perforation of human epithelial carcinoma cell with antibody-conjugated biodegradable microspheres illuminated by a single 80 femtosecond near-infrared laser pulse.

Terakawa M, Tsunoi Y, Mitsuhashi T - Int J Nanomedicine (2012)

Fluorescence (A and C) and phase contrast (B and D) images of A431 cells perforated by using antibody-conjugated polylactic acid spheres irradiated by a single fs laser pulse at 1.06 J/cm2 in the presence of fluorescein isothiocyanate-dextran (A and B) and Alexa Fluor-labeled small interfering RNA (C and D).Note: Dashed circles (300 μm diameter) indicate the laser irradiated area. Abbreviation: fs, femtosecond.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3368514&req=5

f4-ijn-7-2653: Fluorescence (A and C) and phase contrast (B and D) images of A431 cells perforated by using antibody-conjugated polylactic acid spheres irradiated by a single fs laser pulse at 1.06 J/cm2 in the presence of fluorescein isothiocyanate-dextran (A and B) and Alexa Fluor-labeled small interfering RNA (C and D).Note: Dashed circles (300 μm diameter) indicate the laser irradiated area. Abbreviation: fs, femtosecond.
Mentions: Figure 4 shows fluorescence images (A and C) and phase contrast images (B and D) of the cells after the 80 fs laser illumination at the laser fluence of 1.06 J/cm2, corresponding to the peak intensity of 1.29 × 1014 W/cm2 under the sphere. The corresponding incident energy to the fs laser illuminated area (300 μm in diameter) is 0.75 mJ. Figure 4A and B show experimental results obtained with the FITC-dextran, while Figure 4C and D show those obtained with the siRNA. The average number of cells in the fs laser illuminated area was 221. Many cells in the illuminated area showed fluorescence, demonstrating the increase in the cell membrane permeability. The average perforation efficiency, which was defined as the fraction of cells in the irradiated area that took up exogenous molecules, evaluated by using FITC-dextran at this laser fluence, was 26.4% ± 7.5% (N = 5), while that evaluated by using siRNA was 34.9% ± 6.5% (N = 7). No statistically significant difference was observed in the perforation efficiencies with FITC-dextran and siRNA (P > 0.05 in nonparametric Mann-Whitney test). The perforation efficiency obtained by using PLA spheres is comparable to that shown in a previous study using PS sphere,23 demonstrating the effectiveness and applicability of biodegradable polymer for the perforation. As shown in Figure 2H, the PLA sphere of 2000 nm diameter works as a microlens and the focused intensity (far field) is kept high for distances longer than a few micrometers under the sphere, reaching a peak value at 870 nm under the sphere. The distance between the antibody-conjugated sphere and the cell membrane is several tens of nanometers. The long-focused zone of the sphere conjugated to the top surface of cell membrane is considered to be in the cytoplasm. Therefore, it is highly probable that the mechanism for perforation is not only due to the ablation of cell membrane.

Bottom Line: A polylactic acid (PLA) sphere, a biodegradable polymer, was used.Fluorescein isothiocyanate (FITC)-dextran and short interfering RNA were delivered into many human epithelial carcinoma cells (A431 cells) by applying a single 80 fs laser pulse in the presence of antibody-conjugated PLA microspheres.Perforation by biodegradable spheres compared with other particles has the potential to be a much safer phototherapy and drug delivery method for patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Electronics and Electrical Engineering, Keio University, Yokohama, Kanagawa, Japan. terakawa@elec.keio.ac.jp

ABSTRACT
Pulsed laser interaction with small metallic and dielectric particles has been receiving attention as a method of drug delivery to many cells. However, most of the particles are attended by many risks, which are mainly dependent upon particle size. Unlike other widely used particles, biodegradable particles have advantages of being broken down and eliminated by innate metabolic processes. In this paper, the perforation of cell membrane by a focused spot with transparent biodegradable microspheres excited by a single 800 nm, 80 fs laser pulse is demonstrated. A polylactic acid (PLA) sphere, a biodegradable polymer, was used. Fluorescein isothiocyanate (FITC)-dextran and short interfering RNA were delivered into many human epithelial carcinoma cells (A431 cells) by applying a single 80 fs laser pulse in the presence of antibody-conjugated PLA microspheres. The focused intensity was also simulated by the three-dimensional finite-difference time-domain method. Perforation by biodegradable spheres compared with other particles has the potential to be a much safer phototherapy and drug delivery method for patients. The present method can open a new avenue, which is considered an efficient adherent for the selective perforation of cells which express the specific antigen on the cell membrane.

Show MeSH
Related in: MedlinePlus